Cells are enveloped by a plasma membrane (“PM”) that consists of a bilayer of phospholipid molecules and several protein molecules. Various phospholipid molecules form the building blocks of the bilayer. The phospholipid molecules are distributed asymmetrically over the two leaflets of the bilayer. Phosphatidylcholine for example is present in both layers, whereas sphingomyeline can be found only in the outer leaflet facing the environment. Aminophospholipids, like phosphatidylserine (“PS”), on the other hand, are predominantly present in the inner leaflet facing the cell's cytosol (Zwaal and Schroit, Blood 89:1121-32 (1997)). Aminophospholipid translocases transport PS from the outer to the inner layer, or leaflet, of the plasma membrane to create an asymmetric distribution of PS. The asymmetric architecture of the PM is a feature of living cells. They expend energy to generate and maintain the uneven distribution of the phospholipid species in their PM's.
A cell can change the phospholipid architecture of its PM under certain circumstances, which lead to activation and perturbation of the cell. Programmed cell death (“PCD”) is associated with the appearance of PS in the outer leaflet of the PM (Fadok et al., J. Immunol. 148:2207-16 (1992)). On the basis of morphology and biochemistry, at least four types of PCD have been identified: (1) apoptosis, (2) apoptosis-like PCD, (3) necrosis-like PCD, and necrosis. Each type is accompanied by a change in the asymmetry of the PM characterized by exposure of PS to the outer layer of the cell surface. PS exposure at the outer layer of the PM is a good indication of a variety of activated and perturbed states of a cell. PS exposure, however, is not exclusively associated with cellular processes culminating in cell death. Transient and reversible PS exposure has been reported for several cell types, including activated B cells, undifferentiated muscle cells prone to form syncytium, chlamydia infected cells, endothelial cells of tumour vasculature (U.S. Pat. No. 6,312,694), and engulfing macrophages (Kenis et al. J. Biol. Chem. 2004 279: 52623-9). In addition, several cellular processes and conditions have been found that are associated with an expression of PS at the outer leaflet of the PM. These include platelet activation, red blood cell ageing, stimulation of the immune system, muscle cell syncytium formation, new blood vessel formation in tumors (U.S. Pat. No. 6,312,694), and tumor growth (Rao et al., Thromb. Res. 67:517-31 (1992)).
In addition, cells can dissipate portions of themselves from their surface resulting in membrane encapsulated microparticles. These microparticles have aminophospholipids such as PS exposed at the outer layer of the membranes. These microparticles have been associated with diseases like infection, AIDS, atherosclerosis. Therefore, aminophospholipids at the cell surface are indicators of a variety of activated and perturbed states of a cell. Moreover, microparticles that exhibit exposed aminophospholipids reflect distant cell activation and perturbation. Hence, phospholipids at the surface of a PM constitute attractive targets for a variety of purposes including research, diagnosis, prevention and treatment of diseases. Preferably, PS in the outer leaflet of a PM constitutes a target for research, diagnosis, prevention and treatment of diseases.
Pharmacological and genetic treatments of diseases are based on the delivery of pharmacologically active compounds to diseased cells where the compounds act preferably intracellularly. Current therapeutic treatments employ systemic delivery of a drug, where the drug circulates through the entire body before reaching its desired target. This method of drug delivery results in systemic dilution of the compound. As a result higher concentrations of the drug are required to achieve a therapeutic efficacy. This is associated with undesired toxic side-effects and increased costs of drugs.
Solutions to these problems are provided by targeted drug delivery systems. The targeting agent, which is coupled to the drugs directly or indirectly, guides the drugs to the diseased cells where they accumulate.
Recently we described annexins, derivatives thereof and annexin-Cys variants as targeting and cell-entry agents and their uses for therapeutic and diagnostic applications (WO 2006/003488, published 12 Jan. 2006). The predominant target of annexins is phosphatidylserine (PS), which is exposed by cells that execute programmed cell death or are submitted to stress such as metabolic stress. The annexins, derivates and annexin-Cys variants as described in WO 2006/003488 bind to cell surface exposed PS and are subsequently internalized. The internalization results in a depletion of surface-bound annexins. This phenomenon disfavours the use of annexins in therapeutic and diagnostic procedures that employ the concept of pretargeting.
Pretargeting is a strategy of targeting a reporting compound for diagnostic purposes and/or a drug for therapeutic purposes to the diseased tissue in a multi-step procedure in order to reduce the background signal and the systemic toxic burden respectively. The pretargeting concept employs two compounds A and B which have a high affinity for each other. Compound A encompasses the targeting function and compound B contains the reporter and/or therapeutic function. Firstly, compound A with the targeting function is administered to the subject. After a certain period of time when the circulating compound A is cleared sufficiently, compound B with the reporter and/or therapeutic drug is given to the subject. The latter compound will accumulate at sites where compound A is retained due to its targeting function. This strategy reduces the amount of compound B that needs to be administered in order to obtain the desired effect. Moreover, it will circumvent background signals and undesired toxic side-effects that are related to compound A if the reporter and/or therapeutic drug were directly coupled to compound A.
Examples of combinations of A and B compounds that have high affinity for each other and that are suitable for pretargeting include the streptavidin/avidin and biotin combination, combination of complementary DNA and RNA oligonucleotides, complementary DNA and RNA analogs such as morpholinos (synthetic oligonucleotide analogues containing morpholino-phosphorodiimidate chains instead of deoxyribose-phosphodiester chains), peptide nucleic acids (synthetic oligonucleotide analogues containing N-aminoethyl-glycine chains instead of deoxyribose-phosphodiester chains, PNA) and aptamers (specifically binding oligonucleotides or oligopeptides), the antibody and hapten combination, and the receptor and ligand combination. These combinations have been used in delivery of radionuclides for imaging and therapy of cancer through the pretargeting strategy (Sharkey et al, Clin. Cancer Res. 2005, 11:7109-21).
The prerequisite for successful implementation of the pretargeting strategy is the accessibility of compound A for compound B. Internalization of compound A by the target cell would reduce the efficacy of this strategy.
Patent application WO 2006/003488, describes that annexins, derivatives thereof, and annexin-Cys variants are internalized by cells that expose PS at their surface. The mechanism of internalization is based on the formation of annexin-trimers and the organization of the annexin-trimers in large 2-dimensional networks (Kenis et al. J. Biol. Chem. 2004 279: 52623-9). This mechanism, thus, diminishes the efficient use of annexin, derivatives thereof, and annexin-Cys variants as targeting parts of compound A in pretargeting strategies.
Mira et al., J. Biol. Chem. 1997, 272: 10474-82, describe annexin mutants M1-M4, which affect Ca2+ binding and the effect thereof on inhibition of cytosolic phospholipase A2.